The interaction of proteins of Sendai virus, influenza virus, and vesicular stomatitis virus with host cell membranes will be studied, concentrating on the interactions occurring on the cytoplasmic surface of the membrane. Three approaches have been developed during the previous grant period for this purpose. First, the envelope of radiolabeled Sendai virus is allowed to fuse with erythrocyte membranes. Inside-out vesicles are then prepared from the erythrocyte membrane, exposing the internal virion proteins to experimental manipulation. Second, virus-infected cells are allowed to attach to polycationic polyacrylamide beads; the cells are lysed, and the plasma membrane is left attached to the bead exposing its cytoplasmic surface. Finally, purified viral proteins will be incorporated into lipid vesicles (liposomes) assembled in vitro. The problems to be studied are those related to virus maturation by budding from the host cell membrane, virus penetration by fusion with the host membrane, and interactions occurring on the internal surface of the virus envelope. The association of viral proteins with host membranes before budding will be studied by pulse chase experiments in vivo. The associations among viral proteins will be studied by selective elution techniques and by chemical cross-linking of labeled viral proteins in the host membrane. Following virus envelope fusion with host cell membranes, the viral nucleocapsid remains attached to the membrane. We will investigate whether this binding influences primary transcription, as would have been predicted from previous studies in other laboratories, and whether the nucleocapsid is released from the membrane during transcription. We will also study the kinetics of the envelope fusion process in a quantitative manner to determine the influence of the state of the host cell membrane on the fusion process. Finally, the membrane interactions of viral proteins in liposomes will be studied by biophysical techniques of fluorescence spectroscopy and spin label electron spin resonance and by chemical cross-linking techniques. These experiments will address the influence of the lipid bilayer on the conformation of viral glycoproteins, interactions among viral proteins on the internal surface of the membrane, and the mechanism of transmembrane communication.